PROJECT SUMMARY Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain in the absence of other identified etiologies for the symptom. IC/BPS patients also frequently have voiding dysfunction such as increased urinary frequency and urgency. This urologic condition is significant and severely affects quality of life. Its financial burden on the U.S. healthcare system is tremendous. Since the etiology of IC/BPS remains elusive, current treatments are largely empirical, often dissatisfactory, and vary in efficacy. Therefore, effort to develop new therapies for IC/BPS is greatly needed. Prior studies have suggested that altered Toll-like receptor (TLR) 4 activation may contribute significantly to the initiation and maintenance of heightened pain states in chronic pain conditions. TLR4 is expressed in numerous cell types including neuronal and non-neuronal cells in the central nervous system as well as immune (e.g. monocytes/macrophages) and other somatic cells (e.g. bladder epithelial cells) in the periphery. Once activated, TLR4 mediates proinflammatory cytokine production, leading to enhanced central and peripheral immune signaling and subsequent exaggerated nociceptive transmission. While the effect of TLR4 inhibition on chronic pain relief has been demonstrated in multiple animal models (e.g. inflammatory and neuropathic pain), this has not been explored in cystitis pain seen in IC/BPS patients. Recently, we identified a major role of altered TLR4/TLR2 activations in IC/BPS. Particularly, we observed that increased production of TLR4-mediated proinflammatory cytokines by peripheral blood mononuclear cells is significantly associated with greater pain severity and the extent of comorbid pain in IC/BPS patients. Consistent with our clinical findings, we found that cystitis pain is associated with elevated systemic and central TLR4/TLR2 inflammatory responses in our validated preclinical transgenic IC/BPS-like animal model (URO-OVA). We hypothesize that altered TLR4/TLR2 activations are responsible for IC/BPS pain and blocking TLR4 and/or TLR2 activation can reverse cystitis pain in IC/BPS patients. To test our hypothesis, we will use the URO-OVA model to conduct three novel and integrative Specific Aims: 1) Identify the states of peripheral and central TLR4/TLR2 activations in cystitis pain in the URO-OVA model; 2) Quantify systemic TLR4 inhibition versus chronic cystitis pain relief to determine the optimum level of systemic TLR4 inhibition for maximum pain relief in the URO-OVA model; 3) Develop TLR4 inhibition combination therapies for cystitis pain in the URO-OVA model. We anticipate that our study will provide new insights for the mechanisms of cystitis pain and a basic research platform for future development of clinical strategies for treatment of IC/BPS pain. Knowledge gained from this study will be useful for ultimately successful clinical care of IC/BPS patients.